【摘要】 目的:研究滤纸干血样苯丙氨酸和葡萄糖-6-磷酸脱氢酶微量连续流式荧光法联合测定实验方法,并评价其在新生儿苯丙酮尿症和葡萄糖-6-磷酸脱氢酶缺乏症筛查中的应用价值。方法:直径3.2 mm的新生儿滤纸干血样经萃取后用微量连续流动荧光分析仪同时测定苯丙氨酸和葡萄糖-6-磷酸脱氢酶浓度,确定苯丙酮尿症和葡萄糖-6-磷酸脱氢酶缺乏症的筛查阳性临界值。结果:在所建立的标本保存和分析条件下,新生儿滤纸干血样经30～120 min萃取后,苯丙氨酸在0～20mg/dl、葡萄糖-6-磷酸脱氢酶在0～150μM NADPH浓度范围内线性良好,苯丙氨酸和葡萄糖-6-磷酸脱氢酶最低检出限分别为0.2 mg/dl和2.0μM NADPH。苯丙氨酸高值和低值质控滤纸干血样的批内变异度分别为4.1%和5.3%,批间变异度分别为5.0%和5.6%;葡萄糖-6-磷酸脱氢酶批内变异度分别为4.9%和5.4%,批间变异度分别为5.1%和5.9%,测定平均回收率分别为98.2%和99.2%,并与PerkinElmer公司PHE和G6PD荧光定量法测定结果高度相关。50009例新生儿滤纸干血样PHE和G6PD呈非正态分布,用95%百分位数法确定筛查新生儿苯丙酮尿症的临界值为PHE>3.9 mg/dl为阳性,筛查新生儿G6PD缺乏症的临界值为G6PD<42μM NADPH为阳性,苯丙酮尿症和葡萄糖-6-磷酸脱氢酶缺乏症筛查阳性率分别为0.064%和0.352%,发病率分别为0.016%和0.042%。结论:微量连续流式荧光分析法可同时检测新生儿滤纸干血样苯丙氨酸和葡萄糖-6-磷酸脱氢酶含量,且灵敏度、特异性、重复性好,为筛查新生儿苯丙酮尿症和葡萄糖-6-磷酸脱氢酶缺乏症提供了简便、快速、费用低的方法。更多还原

【Abstract】 Objective:To develop a micro-continuous flow fluorescence for Simultaneous determination of Phenylalanine and Glucose-6-Phosphate dehydrogenase(G6PD) in dry blood specimens of filter paper and to evaluate its application in newborn screening test for phenylketonuria(PHE) and Glucose-6-Phosphate dehydrogenase(G6PD)deficiency.Methods:A neonatal dry blood specimen of filter paper whose diameter was 3.2 mm was extracted first,then measured its Phenylalanine and Glucose-6-Phosphate dehydrogenase concentration simultaneously with micro-continuous flow fluorescence analyzer to determine cut-off value in screening of PHE and G6PD deficiency.Results:Under established preservation and analysis condition,after 30 ~ 120min extraction of neonatal dry blood specimens of filter paper,there was a good linear correlation when phenylalanine was in 0 ~ 20mg/dl,G6PD was in 0~150 μ M NADPH.For PHE in quality control dried blood spot of filter paper,the intra-assay variation of high-value and low-value were 4.1% and 5.3% and the inter-assay variation were 5.0% and 5.6% respectively.For G6PD,the intra-assay variation were 4.9% and 5.4% and inter-assay variation were 5.1% and 5.9% respectively.The average recoveries were 98.2% and 99.2% respectively,that results were highly correlated with the results using PerkinElmer’s PHE and G6PD fluorescence quantitative method.There was a non-normal distribution of PHE and G6PD values from 50 009 dry blood specimens of filter paper.The positive critical value of PHE was determined greater than 3.9mg/dl with 95% percentile for newborn screening for phenylketonuria,and the positive ciritical value of G6PD was less than 42 μM NADPH for glucose-6-phosphate dehydrogenase deficiency,their both screening positive rates were 0.064% and 0.352%,and the incidence rates were 0.016% and the 0.042%,respectively.Conclusion:Micro-continuous flow fluorescence analysis can detect phenylalanine and glucose-6-phosphate dehydrogenase levels in neonatal dry blood specimens of filter paper at same time,and have good sensitivity,specificity and reproducibility.It is a simple,fast,low-cost method of newborn screening for phenylketonuria and glucose-6-phosphate dehydrogenase deficiency.更多还原